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Background: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence., Methods: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls., Results: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 10 2 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 10 1 CFU/mL of river water and 2 × 10 3 CFU/gm of paddy field soil., Conclusions: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices., (© 2024. The Author(s).)

Background: Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection of B. pseudomallei is required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences of B. pseudomallei and development of quantitative polymerase chain reaction (qPCR) assay., Methods: A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei., Results: In this study, an 80-nucleotide-long B. pseudomallei-specific region within the gene HrpB1 was identified by computational analysis. The developed HrpB1-based qPCR assay was highly specific for B. pseudomallei detection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with the HrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 10 2 CFU/mL for human blood and urine, 5 × 10 1 CFU/mL in river water, and 2 × 10 3 CFU/gm in paddy field soil., Conclusion: The results of the study showed the applicability of a novel HrpB1-based qPCR assay for sensitive and specific detection of B. pseudomallei in diverse clinical and environmental samples., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus , cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden. Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella . Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection. Methodology. We purified recombinant rOmp28 with Ni-NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella . Checkerboard sandwich ELISA and P/N ratio (optical density of 'P' positive test sample to 'N' negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella . Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml -1 ) and rOmp28-derived mice IgG (detection antibody at 100 µg ml -1 ) with a detection range of 10 2 to 10 8 cells ml -1 and a limit of detection at 10 2 cells ml -1 . A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria. Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock., (© 2021. The Author(s).)

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10-100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 10 2 to 10 8 cells mL -1 and limit of detection at 10 3 cells mL -1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL -1 ) and mice IgG (detection antibody, 50 µg mL -1 ) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 10 2 cells mL -1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (K D ) and Maximum Binding Capacity (B max ) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Q fever is an important zoonotic disease caused by the bacterium Coxiella burnetii . The agent is considered as a potential agent for bioterrorism because of its low infectious dose, aerial route of transmission, resistance to drying, and many commonly used disinfectants. Humans are largely infected by the inhalation of aerosols that are contaminated with parturition products of infected animals as well as by the consumption of unpasteurized milk products. Thus, rapid and accurate detection of C. burnetii in shedders, especially those that are asymptomatic, is important for early warning, which allows controlling its spread among animals and animal-to-human transmission. In the present study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was developed to confirm the presence of IS 1111a gene of C. burnetii in sheep vaginal swabs. The sensitivity of this assay was found to be very comparable to the quantitative PCR (qPCR) assay, which could detect three copies of the gene, which corresponds to a single cell of C. burnetii . The applicability of the colorimetric LAMP assay in the disease diagnosis was assessed by evaluating 145 vaginal swab samples collected from the sheep breeding farms with a history of stillbirth and repeated abortions. Compared to qPCR, colorimetric LAMP had a sensitivity of 93.75% (CI, 69.77-99.84%) and specificity of 100% (CI, 97.20-100%), with a positive (PPV) and negative predictive value (NPV) of 100 and 99.24%, respectively. A very high level of agreement was observed between both colorimetric LAMP and reference qPCR assay. The colorimetric LAMP assay reported here is a rapid and simple test without extensive sample preparation and has a short turnaround time of <45 min. To the best of our understanding, it is the very first study describing the use of colorimetric LAMP assay that detects C. burnetii in vaginal swab samples with minimal sample processing for DNA extraction., (Copyright © 2020 Sheikh, Kumar, Sharma, Bhagyawant and Thavaselvam.)

Brucellosis is a zoonotic disease caused by various species of the genus Brucella. The control of this disease mainly depends on its accurate and early diagnosis. Culture methods employed for diagnosis are time consuming and require well equipped biosafety level 3 laboratories and hence serological tests are favored alternative for brucellosis diagnosis. At present serological diagnosis is based on LPS (lipopolysaccharide) which is less specific as it shows cross reactivity with other gram-negative bacteria. There is a need to develop serological diagnostic assay based on purified recombinant antigen of Brucella. T4SS (Type IV Secretion System) is an important virulent factor of Brucella and required for infection suggesting their expression in vivo and can be targeted as serological marker for infection. To test this concept, the present study is designed to clone, express and purify virB10 gene of Brucella T4SS under denaturing conditions and to evaluate its use as serological marker of Brucella infection. The immunoreactivity of this recombinant antigen was checked with antisera collected after experimental infection in Balb/C mice with B. melitensis 16M, BR31 (human clinical isolate) and Y. enterocolitica O:9. The recombinant protein was also tested against a panel of 46 bovine sera samples collected from Leh, India. Antibody response against VirB10 was detected in experimental and natural host suggesting that it can be explored as potential target for serodiagnosis of Brucella infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay)., Method: Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23)., Result: The rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student's t test was performed on the data., Conclusion: Omp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (K D ) and maximum binding capacity of analyte (B max ) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (K D ) and 81.02m° (B max ) with probe 1 and 54.9pM and 55.29m° (B max ), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis., Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS 711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization., Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M., Interpretation & Conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis.

Melioidosis is an emerging infectious disease in India and caused by gram-negative, soil saprophyte bacteria Burkholderia pseudomallei. This disease is endemic in Southeast Asia and northern Australia, and sporadic cases of melioidosis are also reported from southern states of India. The present study reports the cloning, expression, and purification of recombinant protein outer membrane protein A (OmpA) of B. pseudomallei and its evaluation in indirect enzyme-linked immunosorbent assay (ELISA) format with 87 serum samples collected from Manipal, Karnataka, India. Twenty-three samples from culture confirmed cases (n=23) of melioidosis, 25 serum samples from patients of other febrile illness and pyrexia of unknown origin (n=25), and 39 serum samples from healthy blood donors (n=39) from Kasturba Medical College, Manipal, were tested in this assay format. The assay showed sensitivity of 82.6% and specificity of 93.75%. The recombinant OmpA based indirect ELISA will be a useful tool for serodiagnosis of melioidosis in large scale rapid screening of clinical samples., (Copyright © 2015 Elsevier Inc. All rights reserved.)